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Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.

Six decades ago, Friedenstein and coworkers published a series of seminal papers identifying a cell population in bone marrow with osteogenic potential, now referred to as mesenchymal stem cells (MSCs). This work was also instrumental in establishing the identity of hematopoietic stem cell and the identification of skeletal stem/progenitor cell (SSPC) populations in various skeletal compartments. In recognition of the centenary year of Friedenstein's birth, I review key aspects of his work and discuss the evolving concept of the MSC and its various euphemisms indorsed by changing paradigms in the field. I also discuss the recent emphasis on MSC stromal quality attributes and how emerging data demonstrating a mechanistic link between stromal and stem/progenitor functions bring renewed relevance to Friedenstein's contributions and much needed unity to the field. [ABSTRACT FROM AUTHOR]

In periodontitis, gingival fibroblasts (GF) appear to produce a multitude of paracrine factors. However, the influence of GF-derived soluble factors on osteoclastogenesis remains unclear. In this case study, production of paracrine factors by GF was assessed under inflammatory and non-inflammatory conditions, as well as their effect on osteoclastogenesis. Human primary GF were cultured in a transwell system and primed with a cocktail of IL-1β, IL-6 and TNF-α to mimic inflammation. GF were co-cultured directly and indirectly with human peripheral blood mononuclear cells (PBMC). Cytokines and chemokines in supernatants (flow cytometry based multiplex assay), osteoclastogenesis (TRAcP staining) and gene expression (qPCR) were quantified on days 7 and 21.Results from this case study showed that GF communicated via soluble factors with PBMC resulting in a two-fold induction of osteoclasts. Reversely, PBMC induced gene expression of IL-6, OPG and MCP-1 by GF. Remarkably, after priming of GF with cytokines, this communication was impaired and resulted in fewer osteoclasts. This could be partly explained by an increase in IL-10 expression and a decrease in MCP-1 expression. Intriguingly, the short priming of GF resulted in significantly higher expression of inflammatory cytokines that was sustained at both 7 and 21 days.GF appear to produce paracrine factors capable of stimulating osteoclastogenesis in the absence of physical cell-cell interactions. GF cultured in the presence of PBMC or osteoclasts had a remarkably inflammatory phenotype. Given profound expression of both pro- and anti-inflammatory cytokines after the inflammatory stimulus, it is probably the effector hierarchy that leads to fewer osteoclasts.

Immune cells infiltrate the peripheral nervous system (PNS) after injury and with autoimmunity, but their net effect is divergent. After injury, immune cells are reparative, while in inflammatory neuropathies (e.g., Guillain Barré Syndrome and chronic inflammatory demyelinating polyneuropathy), immune cells are proinflammatory and promote autoimmune demyelination. An understanding of immune cell phenotypes that distinguish these conditions may, therefore, reveal new therapeutic targets for switching immune cells from an inflammatory role to a reparative state. In an autoimmune regulator (Aire)-deficient mouse model of inflammatory neuropathy, we used single-cell RNA sequencing of sciatic nerves to discover a transcriptionally heterogeneous cellular landscape, including multiple myeloid, innate lymphoid, and lymphoid cell types. Analysis of cell-cell ligand-receptor interactions uncovered a macrophage-mediated tumor necrosis factor-α (TNF-α) signaling axis that is induced by interferon-γ and required for initiation of autoimmune demyelination. Developmental trajectory visualization suggested that TNF-α signaling is associated with metabolic reprogramming of macrophages and polarization of macrophages from a reparative state in injury to a pathogenic, inflammatory state in autoimmunity. Autocrine TNF-α signaling induced macrophage expression of multiple genes (Clec4e, Marcksl1, Cxcl1, and Cxcl10) important in immune cell activation and recruitment. Genetic and antibody-based blockade of TNF-α/TNF-α signaling ameliorated clinical neuropathy, peripheral nerve infiltration, and demyelination, which provides preclinical evidence that the TNF-α axis may be effectively targeted to resolve inflammatory neuropathies.

As an incretin hormone, glucagon-like peptide 1 (GLP-1) lowers blood glucose levels by enhancing glucose-stimulated insulin secretion from pancreatic beta-cells. Therapies targeting the GLP-1 receptor (GLP-1R) use the classical incretin model as a physiological framework in which GLP-1 secreted from enteroendocrine L-cells acts on the beta-cell GLP-1R. However, this model has come into question, as evidence demonstrating local, intra-islet GLP-1 production has advanced the competing hypothesis that the incretin activity of GLP-1 may reflect paracrine signaling of GLP-1 from alpha-cells on GLP-1Rs on beta-cells. Additionally, recent studies suggest that alpha-cell-derived glucagon can serve as an additional, albeit less potent, ligand for the beta-cell GLP-1R, thereby expanding the role of alpha-cells beyond that of a counterregulatory cell type. Efforts to understand the role of the alpha-cell in the regulation of islet function have revealed both transcriptional and functional heterogeneity within the alpha-cell population. Further analysis of this heterogeneity suggests that functionally distinct alpha-cell subpopulations display alterations in islet hormone profile. Thus, the role of the alpha-cell in glucose homeostasis has evolved in recent years, such that alpha-cell to beta-cell communication now presents a critical axis regulating the functional capacity of beta-cells. Herein, we describe and integrate recent advances in our understanding of the impact of alpha-cell paracrine signaling on insulin secretory dynamics and how this intra-islet crosstalk more broadly contributes to whole-body glucose regulation in health and under metabolic stress. Moreover, we explore how these conceptual changes in our understanding of intra-islet GLP-1 biology may impact our understanding of the mechanisms of incretin-based therapeutics.

AimsStem cell therapy has shown promise for treating myocardial infarction via re-muscularization and paracrine signalling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischaemic conditions in the infarcted heart under these two different delivery approaches remains unclear. Here, we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level.Methods and resultsNHP iPSC-CMs in both monolayer culture (2D) and engineered heart tissue (EHT) (3D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3D EHT model was more sensitive to ischaemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signalling.ConclusionBy exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischaemic injury.

Despite its abundance in the environment, iron is poorly bioavailable and subject to strict conservation and internal recycling by most organisms. In vertebrates, the stability of iron concentration in plasma and extracellular fluid, and the total body iron content are maintained by the interaction of the iron-regulatory peptide hormone hepcidin with its receptor and cellular iron exporter ferroportin (SLC40a1). Ferroportin exports iron from duodenal enterocytes that absorb dietary iron, from iron-recycling macrophages in the spleen and the liver, and from iron-storing hepatocytes. Hepcidin blocks iron export through ferroportin, causing hypoferremia. During iron deficiency or after hemorrhage, hepcidin decreases to allow iron delivery to plasma through ferroportin, thus promoting compensatory erythropoiesis. As a host defense mediator, hepcidin increases in response to infection and inflammation, blocking iron delivery through ferroportin to blood plasma, thus limiting iron availability to invading microbes. Genetic diseases that decrease hepcidin synthesis or disrupt hepcidin binding to ferroportin cause the iron overload disorder hereditary hemochromatosis. The opposite phenotype, iron restriction or iron deficiency, can result from genetic or inflammatory overproduction of hepcidin.

Targeted therapies for gastrointestinal stromal tumor (GIST) are modestly effective, but GIST cannot be cured with single agent tyrosine kinase inhibitors. In this study, we sought to identify new therapeutic targets in GIST by investigating the tumor microenvironment. Here, we identified a paracrine signaling network by which cancer-associated fibroblasts (CAFs) drive GIST growth and metastasis. Specifically, CAFs isolated from human tumors were found to produce high levels of platelet-derived growth factor C (PDGFC), which activated PDGFC-PDGFRA signal transduction in GIST cells that regulated the expression of SLUG, an epithelial-mesenchymal transition (EMT) transcription factor and downstream target of PDGFRA signaling. Together, this paracrine induce signal transduction cascade promoted tumor growth and metastasis in vivo. Moreover, in metastatic GIST patients, SLUG expression positively correlated with tumor size and mitotic index. Given that CAF paracrine signaling modulated GIST biology, we directly targeted CAFs with a dual PI3K/mTOR inhibitor, which synergized with imatinib to increase tumor cell killing and in vivo disease response. Taken together, we identified a previously unappreciated cellular target for GIST therapy in order to improve disease control and cure rates.

BackgroundMicrotia is an inherited condition that results in varying degrees of external ear deformities; the most extreme form is anotia. Effective surgical reconstruction techniques have been developed. However, these usually require multistage procedures and have other inherent disadvantages. Tissue engineering technologies offer new approaches in the field of external ear reconstruction. In this setting, chondrocytes are cultured in the laboratory with the aim of creating bioengineered cartilage matrices. However, cartilage engineering has many challenges, including difficulty in culturing sufficient chondrocytes. To overcome these hurdles, the authors propose a novel model of cartilage engineering that involves co-culturing chondrocytes and adipose-derived stem cells on an allograft adipose-derived extracellular matrix scaffold.MethodsAuricular chondrocytes from porcine ear were characterized. Adipose-derived stem cells were isolated and expanded from human lipoaspirate. Then, the auricular chondrocytes were cultured on the allograft adipose matrix either alone or with the adipose-derived stem cells at different ratios and examined histologically.ResultsCartilage induction was most prominent when the cells were co-cultured on the allograft adipose matrix at a ratio of 1:9 (auricular chondrocyte-to-adipose-derived stem cell ratio). Furthermore, because of the xenogeneic nature of the experiment, the authors were able to determine that the adipose-derived stem cells contributed to chondrogenesis by means of a paracrine stimulation of the chondrocytes.ConclusionsIn this situation, adipose-derived stem cells provide sufficient support to induce the formation of cartilage when the number of auricular chondrocytes available is limited. This novel model of cartilage engineering provides a setting for using the patient's own chondrocytes and adipose tissue to create a customized ear framework that could be further used for surgical reconstruction.

Six decades ago, Friedenstein and coworkers published a series of seminal papers identifying a cell population in bone marrow with osteogenic potential, now referred to as mesenchymal stem cells (MSCs). This work was also instrumental in establishing the identity of hematopoietic stem cell and the identification of skeletal stem/progenitor cell (SSPC) populations in various skeletal compartments. In recognition of the centenary year of Friedenstein’s birth, I review key aspects of his work and discuss the evolving concept of the MSC and its various euphemisms indorsed by changing paradigms in the field. I also discuss the recent emphasis on MSC stromal quality attributes and how emerging data demonstrating a mechanistic link between stromal and stem/progenitor functions bring renewed relevance to Friedenstein’s contributions and much needed unity to the field.

Background & aimsEosinophilic esophagitis (EoE) is an antigen-mediated eosinophilic disease of the esophagus that involves fibroblast activation and progression to fibrostenosis. Cytokines produced by T-helper type 2 cells and transforming growth factor beta 1 (TGFβ1) contribute to the development of EoE, but other cytokines involved in pathogenesis are unknown. We investigate the effects of tumor necrosis factor superfamily member 14 (TNFSF14, also called LIGHT) on fibroblasts in EoE.MethodsWe analyzed publicly available esophageal CD3+ T-cell single-cell sequencing data for expression of LIGHT. Esophageal tissues were obtained from pediatric patients with EoE or control individuals and analyzed by immunostaining. Human primary esophageal fibroblasts were isolated from esophageal biopsy samples of healthy donors or patients with active EoE. Fibroblasts were cultured; incubated with TGFβ1 and/or LIGHT; and analyzed by RNA sequencing, flow cytometry, immunoblots, immunofluorescence, or reverse transcription polymerase chain reaction. Eosinophils were purified from peripheral blood of healthy donors, incubated with interleukin 5, cocultured with fibroblasts, and analyzed by immunohistochemistry.ResultsLIGHT was up-regulated in the esophageal tissues from patients with EoE, compared with control individuals, and expressed by several T-cell populations, including T-helper type 2 cells. TNF receptor superfamily member 14 (TNFRSF14, also called HVEM) and lymphotoxin beta receptor are receptors for LIGHT that were expressed by fibroblasts from healthy donors or patients with active EoE. Stimulation of esophageal fibroblasts with LIGHT induced inflammatory gene transcription, whereas stimulation with TGFβ1 induced transcription of genes associated with a myofibroblast phenotype. Stimulation of fibroblasts with TGFβ1 increased expression of HVEM; subsequent stimulation with LIGHT resulted in their differentiation into cells that express markers of myofibroblasts and inflammatory chemokines and cytokines. Eosinophils tethered to esophageal fibroblasts after LIGHT stimulation via intercellular adhesion molecule-1.ConclusionsT cells in esophageal tissues from patients with EoE express increased levels of LIGHT compared with control individuals, which induces differentiation of fibroblasts into cells with inflammatory characteristics. TGFβ1 increases fibroblast expression of HVEM, a receptor for LIGHT. LIGHT mediates interactions between esophageal fibroblasts and eosinophils via ICAM1. This pathway might be targeted for the treatment of EoE.

Pancreatic beta cells are the only cell type in our body capable of producing and secreting insulin to instruct the insulin-sensitive cells and tissues of our bodies to absorb nutrients after a meal. Accurate control of insulin release is of critical importance; too little insulin leads to diabetes, while an excess of insulin can cause potentially fatal hypoglycaemia. Yet, the pancreas of most people will control insulin secretion safely and effectively over decades and in response to glucose excursions driven by tens of thousands of meals. Because we only become aware of the important contributions of the pancreas when it fails to maintain glucose homeostasis, it is easy to forget just how well insulin release from a healthy pancreas is matched to insulin need to ensure stable blood glucose levels. Beta cells achieve this feat by extensive crosstalk with the rest of the endocrine cell types in the islet, notably the glucagon-producing alpha cells and somatostatin-producing delta cells. Here I will review the important paracrine contributions that each of these cells makes to the stimulation and subsequent inhibition of insulin release in response to a transient nutrient stimulation, and make the case that a breakdown of this local crosstalk contributes to the pathophysiology of diabetes. Graphical abstract.

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.

Stromal-epithelial interactions dictate cancer progression and therapeutic response. Prostate cancer (PCa) cells were identified to secrete greater concentration of mitochondrial DNA (mtDNA) compared to noncancer epithelia. Based on the recognized coevolution of cancer-associated fibroblasts (CAF) with tumor progression, we tested the role of cancer-derived mtDNA in a mechanism of paracrine signaling. We found that prostatic CAF expressed DEC205, which was not expressed by normal tissue-associated fibroblasts. DEC205 is a transmembrane protein that bound mtDNA and contributed to pattern recognition by Toll-like receptor 9 (TLR9). Complement C3 was the dominant gene targeted by TLR9-induced NF-κB signaling in CAF. The subsequent maturation complement C3 maturation to anaphylatoxin C3a was dependent on PCa epithelial inhibition of catalase in CAF. In a syngeneic tissue recombination model of PCa and associated fibroblast, the antagonism of the C3a receptor and the fibroblastic knockout of TLR9 similarly resulted in immune suppression with a significant reduction in tumor progression, compared to saline-treated tumors associated with wild-type prostatic fibroblasts. Interestingly, docetaxel, a common therapy for advanced PCa, further promoted mtDNA secretion in cultured epithelia, mice, and PCa patients. The antiapoptotic signaling downstream of anaphylatoxin C3a signaling in tumor cells contributed to docetaxel resistance. The inhibition of C3a receptor sensitized PCa epithelia to docetaxel in a synergistic manner. Tumor models of human PCa epithelia with CAF expanded similarly in mice in the presence or absence of docetaxel. The combination therapy of docetaxel and C3 receptor antagonist disrupted the mtDNA/C3a paracrine loop and restored docetaxel sensitivity.

Background: In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis are not well understood. To address these questions, we performed a comparative analysis of publicly available single-cell transcriptomes of healthy adult human DP to 5 other reference tissues: peripheral blood mononuclear cells, bone marrow, adipose tissue, lung, and skin. Results: Our analysis revealed that DP resident cells have a unique gene expression profile when compared to the reference tissues, and that DP fibroblasts are the main cell type contributing to this expression profile. Genes coding for pleiotrophin (PTN) and midkine (MDK), homologous heparin-binding growth-factors, possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP resident cells, including Schwann cells, mesenchymal stem cells and odontoblasts, mediated by PTN and MDK. Conclusions: DP fibroblasts emerge as unappreciated players in DP homeostasis, mainly through their crosstalk with glial cells. These findings suggest that fibroblast-derived growth factors possess major regulatory functions and thus have a potential role as dental therapeutic targets. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) results from the progressive loss of pancreatic beta cells as a result of autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. Here, we show that the SASP in islet cells is transcriptionally controlled by Bromodomain ExtraTerminal (BET) proteins, including Bromodomain containing protein 4 (BRD4). A chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active regulatory regions. BET protein inhibition in NOD islets diminished not only the transcriptional activation and secretion of SASP factors, but also the non-cell autonomous activity. BET protein inhibition also decreased the extent of SASP induction in human islets exposed to DNA damage. The BET protein inhibitor iBET-762 prevented diabetes in NOD mice and also attenuated SASP in islet cells in vivo. Taken together, our findings support a crucial role for BET proteins in the activation of the SASP transcriptional program in islet cells. These studies suggest avenues for preventing T1D by transcriptional inhibition of SASP.

Transplanted stromal cells have demonstrated considerable promise as therapeutic agents in diverse disease settings. Paracrine signaling can be an important mediator of these therapeutic effects at the sites of acute or persistent injury and inflammation. As many stromal cell types, including bone marrow-derived stromal cells (BMSCs), display tissue-specific responses, there is a need to explore their secretory dynamics in the context of tissue and injury type. Paracrine signals are not static, and could encode contextual dynamics in the kinetic changes of the concentrations of the secreted ligands. However, precise measurement of dynamic and context-specific cellular secretory signatures, particularly in adherent cells, remains challenging. Here, by creating an experimental and computational analysis platform, we reconstructed dynamic secretory signatures of cells based on a very limited number of time points. By using this approach, we demonstrate that the secretory signatures of CD133-positive BMSCs are uniquely defined by distinct biological contexts, including signals from injured cardiac cells undergoing oxidative stress, characteristic of cardiac infarction. Furthermore, we show that the mixture of recombinant factors reproducing the dynamics of BMSC-generated secretion can mediate a highly effective rescue of cells injured by oxidative stress and an improved cardiac output. These results support the importance of the dynamic multifactorial paracrine signals in mediating remedial effects of stromal stem cells, and pave the way for stem cell-inspired cell-free treatments of cardiac and other injuries.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

Breast cancer development is associated with increasing tissue stiffness over years. To more accurately mimic the onset of gradual matrix stiffening, which is not feasible with conventional static hydrogels, mammary epithelial cells (MECs) were cultured on methacrylated hyaluronic acid hydrogels whose stiffness can be dynamically modulated from "normal" (3,000 Pascals) via two-stage polymerization. MECs form and remain as spheroids, but begin to lose epithelial characteristics and gain mesenchymal morphology upon matrix stiffening. However, both the degree of matrix stiffening and culture time before stiffening play important roles in regulating this conversion as, in both cases, a subset of mammary spheroids remained insensitive to local matrix stiffness. This conversion depended neither on colony size nor cell density, and MECs did not exhibit "memory" of prior niche when serially cultured through cycles of compliant and stiff matrices. Instead, the transcription factor Twist1, transforming growth factor β (TGFβ), and YAP activation appeared to modulate stiffness-mediated signaling; when stiffness-mediated signals were blocked, collective MEC phenotypes were reduced in favor of single MECs migrating away from spheroids. These data indicate a more complex interplay of time-dependent stiffness signaling, spheroid structure, and soluble cues that regulates MEC plasticity than suggested by previous models.

The cytokine IFN-γ is a critical regulator of immune system development and function. Almost all leukocytes express the receptor for IFN-γ, yet each cell type elicits a different response to this cytokine. Cell type-specific effects of IFN-γ make it difficult to predict the outcomes of the systemic IFN-γ blockade and limit its clinical application, despite many years of research. To better understand the cell-cell interactions and cofactors that specify IFN-γ functions, we focused on the function of IFN-γ on CD8 T cell differentiation. We demonstrated that during bacterial infection, IFN-γ is a dominant paracrine trigger that skews CD8 T cell differentiation toward memory. This skewing is preferentially driven by contact-dependent T cell-T cell (T-T) interactions and the localized IFN-γ secretion among activated CD8 T cells in a unique splenic microenvironment, and is less sensitive to concurrent IFN-γ production by other immune cell populations such as natural killer (NK) cells. Modulation of CD8 T cell differentiation by IFN-γ relies on a nonconventional IFN-γ outcome that occurs specifically within 24 hours following infection. This is driven by IFN-γ costimulation by integrins at T-T synapses, and leads to synergistic phosphorylation of the proximal STAT1 molecule and accelerated IL-2 receptor down-regulation. This study provides evidence of the importance of context-dependent cytokine signaling and gives another example of how cell clusters and the microenvironment drive unique biology.

Chemotherapy and radiation not only trigger cancer cell apoptosis but also damage stromal cells in the tumour microenvironment (TME), inducing a senescence-associated secretory phenotype (SASP) characterized by chronic secretion of diverse soluble factors. Here we report serine protease inhibitor Kazal type I (SPINK1), a SASP factor produced in human stromal cells after genotoxic treatment. DNA damage causes SPINK1 expression by engaging NF-κB and C/EBP, while paracrine SPINK1 promotes cancer cell aggressiveness particularly chemoresistance. Strikingly, SPINK1 reprograms the expression profile of cancer cells, causing prominent epithelial-endothelial transition (EET), a phenotypic switch mediated by EGFR signaling but hitherto rarely reported for a SASP factor. In vivo, SPINK1 is expressed in the stroma of solid tumours and is routinely detectable in peripheral blood of cancer patients after chemotherapy. Our study substantiates SPINK1 as both a targetable SASP factor and a novel noninvasive biomarker of therapeutically damaged TME for disease control and clinical surveillance.

Cancer and other cells residing in the same niche engage various modes of interactions to synchronize and buffer the negative effects of environmental changes. Extracellular microRNAs (miRNAs) have recently been implicated in the intercellular crosstalk. Here we show a mechanistic model involving breast-cancer-secreted, extracellular-vesicle-encapsulated miR-105, which is induced by the oncoprotein MYC in cancer cells and, in turn, activates MYC signalling in cancer-associated fibroblasts (CAFs) to induce a metabolic program. This results in the capacity of CAFs to display different metabolic features in response to changes in the metabolic environment. When nutrients are sufficient, miR-105-reprogrammed CAFs enhance glucose and glutamine metabolism to fuel adjacent cancer cells. When nutrient levels are low and metabolic by-products accumulate, these CAFs detoxify metabolic wastes, including lactic acid and ammonium, by converting them into energy-rich metabolites. Thus, the miR-105-mediated metabolic reprogramming of stromal cells contributes to sustained tumour growth by conditioning the shared metabolic environment.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and remarkable increases in postprandial glucagon-like peptide-1 (GLP-1) secretion. GLP-1 plays a critical role in islet function by potentiating glucose-stimulated insulin secretion; however, the mechanisms remain incompletely defined. Therefore, we applied a murine VSG model to an inducible β cell-specific GLP-1 receptor (GLP-1R) knockout mouse model to investigate the role of the β cell GLP-1R in islet function. Our data show that loss of β cell GLP-1R signaling decreases α cell GLP-1 expression after VSG. Furthermore, we find a β cell GLP-1R-dependent increase in α cell expression of the prohormone convertase required for the production of GLP-1 after VSG. Together, the findings herein reveal two concepts. First, our data support a paracrine role for α cell-derived GLP-1 in the metabolic benefits observed after VSG. Second, we have identified a role for the β cell GLP-1R as a regulator of α cell proglucagon processing.

Cardiovascular regenerative therapies are pursued on both basic and translational levels. Although efficacy and value of cell therapy for myocardial regeneration can be debated, there is a consensus that profound deficits in mechanistic understanding limit advances, optimization, and implementation. In collaboration with the TACTICS (Transnational Alliance for Regenerative Therapies in Cardiovascular Syndromes), this review overviews several pivotal aspects of biological processes impinging on cardiac maintenance, repair, and regeneration. The goal of summarizing current mechanistic understanding is to prompt innovative directions for fundamental studies delineating cellular reparative and regenerative processes. Empowering myocardial regenerative interventions, whether dependent on endogenous processes or exogenously delivered repair agents, ultimately depends on mastering mechanisms and novel strategies that take advantage of rather than being limited by inherent myocardial biology.

Marina crystal minerals (MCM) are a mixture that contains crystallized minerals along with trace elements extracted from seawater. It is a nutritional supplement that is capable of enhancing natural killer (NK) cell activity and increasing T and B cell proliferation in humans post ingestion. However, its effect on dendritic cells (DCs), the cells that bridge innate and adaptive immunity, is not yet known. In this study, we examine the stimulatory effects of MCM on DCs' maturation and function in vitro. Human monocyte-derived DCs were treated with MCM at two different concentrations (10 and 20 µg/mL) for 24 h. Results showed that MCM treatment activated DCs in a dose-dependent fashion. It caused the upregulation of costimulatory molecules CD80, CD86, and HLA-DR, and prompted the production of DC cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1β, and chemokines (monocyte chemotactic protein-1 (MCP-1)) and interferon-gamma-inducible protein-10 (IP-10). In addition, activated DCs primed CD4+ T cells to secrete significant amounts of interferon gamma (IFN-γ), and they also stimulated CD8+ T cells to express higher amounts of CD107a. These results indicate that MCM is a potentially powerful adjuvant, from natural materials, that activates human DCs in vitro and therefore may suggest its possible use in immune-based therapies against cancer and viral infections.

The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis. We identify HGF, acting through the c-Met receptor, as the key polarity-inducing morphogen, which acts to activate β1-integrin-dependent adhesion. HGF and ECM-derived integrin signals co-operate via a c-Src-dependent inhibition of the RhoA-ROCK1 signalling pathway via p190A RhoGAP. This occurred via controlling localization of these signalling pathways to the ECM-abutting surface of cells in 3-Dimensional culture. Thus, stromal derived signals can influence morphogenesis in epithelial cells by controlling activation and localization of cell polarity pathways.

Enteroendocrine cells (EECs) are well-known for their systemic hormonal effects, especially in the regulation of appetite and glycemia. Much less is known about how the products made by EECs regulate their local environment within the intestine. Here, we focus on paracrine interactions between EECs and other intestinal cells as they regulate three essential aspects of intestinal homeostasis and physiology: 1) intestinal stem cell function and proliferation; 2) nutrient absorption; and 3) mucosal barrier function. We also discuss the ability of EECs to express multiple hormones, describe in vitro and in vivo models to study EECs, and consider how EECs are altered in GI disease., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Obesity-induced β cell dysfunction contributes to the onset of type 2 diabetes. Nevertheless, elucidating epigenetic mechanisms underlying islet dysfunction at single cell level remains challenging. Here we profile single-nuclei RNA along with enhancer marks H3K4me1 or H3K27ac in islets from lean or obese mice. Our study identifies distinct gene signatures and enhancer states correlating with β cell dysfunction trajectory. Intriguingly, while many metabolic stress-induced genes exhibit concordant changes in both H3K4me1 and H3K27ac at their enhancers, expression changes of specific subsets are solely attributable to either H3K4me1 or H3K27ac dynamics. Remarkably, a subset of H3K4me1 + H3K27ac - primed enhancers prevalent in lean β cells and occupied by FoxA2 are largely absent after metabolic stress. Lastly, cell-cell communication analysis identified the nerve growth factor (NGF) as protective paracrine signaling for β cells through repressing ER stress. In summary, our findings define the heterogeneous enhancer responses to metabolic challenges in individual β cells., (© 2024. The Author(s).)

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall, diastolic dysfunction, and fibrosis, and is associated with mutations in genes encoding sarcomere proteins. While in vitro studies have used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined the multicellular interactions involved in fibrosis. Using engineered cardiac microtissues (CMTs) composed of HCM-causing MYH7 -variant hiPSC-CMs and wild-type fibroblasts, we observed cell-cell cross-talk leading to increased collagen deposition, tissue stiffening, and decreased contractility dependent on fibroblast proliferation. hiPSC-CM conditioned media and single-nucleus RNA sequencing data suggested that fibroblast proliferation is mediated by paracrine signals from MYH7 -variant cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor tyrosine kinase with erlotinib hydrochloride attenuated stromal activation. Last, HCM-causing MYBPC3 -variant CMTs also demonstrated increased stromal activation and reduced contractility, but with distinct characteristics. Together, these findings establish a paracrine-mediated cross-talk potentially responsible for fibrotic changes observed in HCM.

Introduction: Macrophages play a key role in most of the inflammatory diseases such as Rheumatoid Arthritis (RA), but the mechanism underlying their pathogenesis is still under study. Among stem cells, human dental pulp stem cells (hDPSCs) have attracted attention due to their easy accessibility and immunomodulatory properties, making them a promising adjuvant therapy. In this study, we aimed to evaluate the capacity of hDPSCs to modulate the phenotypes of primary human macrophages. Additionally, we sought to observe the differences induced on macrophages when cultured directly with hDPSCs or through a cell culture insert, mimicking the paracrine communication pathway., Methods: Monocytes, isolated from buffy coats, were differentiated into pro-inflammatory M1 and anti-inflammatory M2 macrophages. Subsequently, they were cultured with hDPSCs either directly or via a cell-culture insert for 48 hours. Finally, they were analyzed for protein, gene expression, cytokines levels and immunofluorescence., Results: In our study, we have demonstrated that, hDPSCs, even without priming, can reduce TNFα levels and enhancing IL-10 release in pro-inflammatory macrophages, both through direct contact and paracrine signaling. Furthermore, we found that their effects are more pronounced when in cell-to-cell contact through the decrease of NF-kB and COX-2 expression and of CD80/PD-L1 colocalization. HDPSCs, when in contact with macrophages, showed enhanced expression of NF-kB, COX-2, ICAM-1, PD-L1, FAS-L, TNFα and IFNγ., Conclusion: We showed that hDPSCs exert immunomodulatory effects on pro-inflammatory macrophages, with cell-to-cell contact yielding a more pronounced outcome compared to paracrine signaling. Our work highlights the immunomodulatory properties of hDPSCs on activated pro-inflammatory macrophages and the potential therapeutic role in inflamed tissue., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Maccaferri, Pisciotta, Carnevale, Salvarani and Pignatti.)

Multiple myeloma (MM) is linked to chronic NF-κB activity in myeloma cells, but this activity is generally considered a cell-autonomous property of the cancer cells. The precise extent of NF-κB activation and the contributions of the physical microenvironment and of cell-to-cell communications remain largely unknown. By quantitative immunofluorescence, we found that NF-κB is mildly and heterogeneously activated in a fraction of MM cells in human BMs, while only a minority of MM cells shows a strong activation. To gain quantitative insights on NF-κB activation in living MM cells, we combined advanced live imaging of endogenous p65 Venus-knocked-in in MM.1S and HS-5 cell lines to model MM and mesenchymal stromal cells (MSCs), cell co-cultures, microfluidics and custom microbioreactors to mimic the 3D-interactions within the bone marrow (BM) microenvironment. We found that i) reciprocal MM-MSC paracrine crosstalk and cell-to-scaffold interactions shape the inflammatory response in the BM; ii) the pro-inflammatory cytokine IL-1β, abundant in MM patients' plasma, activates MSCs, whose paracrine signals are responsible for strong NF-κB activation in a minority of MM cells; iii) IL-1β, but not TNF-α, activates NF-κB in vivo in BM-engrafted MM cells, while its receptor inhibitor Anakinra reduces the global NF-κB activation. We propose that NF-κB activation in the BM of MM patients is mild, restricted to a minority of cells and modulated by the interplay of restraining physical microenvironmental cues and activating IL-1β-dependent stroma-to-MM crosstalk., (© 2024. The Author(s).)

Organ architecture is established during development through intricate cell-cell communication mechanisms, yet the specific signals mediating these communications often remain elusive. Here, we used the anterior pituitary gland that harbors different interdigitated hormone-secreting homotypic cell networks to dissect cell-cell communication mechanisms operating during late development. We show that blocking differentiation of corticotrope cells leads to pituitary hypoplasia with a major effect on somatotrope cells that directly contact corticotropes. Gene knockout of the corticotrope-restricted transcription factor Tpit results in fewer somatotropes, with less secretory granules and a loss of cell polarity, resulting in systemic growth retardation. Single-cell transcriptomic analyses identified FGF1 as a corticotrope-specific Tpit dosage-dependent target gene responsible for these phenotypes. Consistently, genetic ablation of FGF1 in mice phenocopies pituitary hypoplasia and growth impairment observed in Tpit -deficient mice. These findings reveal FGF1 produced by the corticotrope cell network as an essential paracrine signaling molecule participating in pituitary architecture and size., Competing Interests: Competing interests statement:The authors declare no competing interest.

Tumour endothelial cells (TECs) are genetically and phenotypically distinct from their normal, healthy counterparts and provide various pro-tumourigenic effects. This study aimed to investigate the impact of conditioned media (CM) from non-tumourigenic MCF-12A breast epithelial cells as well as from MCF-7 and MDA-MB-231 breast cancer cells on human umbilical vein endothelial cells (HUVECs). Significant increases in cell viability were observed across all breast CM groups compared to controls, with notable differences between the MCF-12A, MCF-7, and MDA-MB-231 groups. Despite increased viability, no significant differences in MCM2 expression, a marker of cell proliferation, were detected. Morphological changes in HUVECs, including elongation, lumen formation, and branching, were more pronounced in breast cancer CM groups, especially in the MDA-MB-231 CM group. qPCR and Western blot analyses showed increased expression of TEC markers such as MDR1, LOX, and TEM8 in HUVECs treated with MCF-12A CM. The MCF-7 CM group significantly enhanced HUVEC migratory activity compared to MCF-12A CM, as evidenced by a scratch assay. These findings underscore distinct angiogenic responses elicited by non-tumourigenic and tumourigenic breast epithelial cells, with tumourigenic cells inducing a hyperactivated angiogenic response. The study highlights the differential effects of breast cancer cell paracrine signalling on endothelial cells and suggests the need for further investigation into TEC markers' role in both physiological and tumour angiogenesis., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Despite of being in different microenvironment, breast cancer cells influence the bone cells and persuade cancer metastasis from breast to bone. Multiple co-culture approaches have been explored to study paracrine signaling between these cells and to study the progression of cancer. However, lack of native tissue microenvironment remains a major bottleneck in existing co-culture technologies. Therefore, in the present study, a tumorigenic and an osteogenic microenvironment have been sutured together to create a multi-cellular environment and has been appraised to study cancer progression in bone tissue. The PCL-polystyrene and PCL-collagen fibrous scaffolds were characterized for tumorigenic and osteogenic potential induction on MDA-MB-231 and MC3T3-E1 cells respectively. Diffusion ability of crystal violet, glucose, and bovine serum albumin across the membrane were used to access the potential paracrine interaction facilitated by device. While in co-cultured condition, MDA-MB-231 cells showed EMT phenotype along with secretion of TNFα and PTHrP which lower down the expression of osteogenic markers including alkaline phosphatase, RUNX2, Osteocalcin and Osteoprotegerin. The cancer progression in bone microenvironment demonstrated the role and necessity of creating multiple tissue microenvironment and its contribution in studying multicellular disease progression and therapeutics., Competing Interests: Declaration of competing interest All the Authors show no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cancer-associated fibroblasts (CAFs) are key contributors to ovarian cancer (OC) progression and therapeutic resistance through dysregulation of the extracellular matrix (ECM). CAFs are a heterogenous population derived from different cell types through activation and reprogramming. Current studies rely on uncharacterized heterogenous primary CAFs or normal fibroblasts that fail to recapitulate CAF-like tumor behavior. Here, we present that conditioned media from ovarian cancer lines leads to an increase in the activated state of fibroblasts demonstrated by functional assays and up-regulation of known CAF-related genes and ECM pathways. Phenotypic and functional characterization demonstrated that the conditioned CAFs expressed a CAF-like phenotype, strengthened proliferation, secretory, contractility, and ECM remodeling properties when compared to resting normal fibroblasts, consistent with an activated fibroblast status. Moreover, conditioned CAFs significantly enhanced drug resistance and tumor progression. Critically, the conditioned CAFs resemble a transcriptional signature with involvement of ECM remodeling. The present study provides mechanistic and functional insights about the activation and reprogramming of CAFs in the ovarian tumor microenvironment mediated by non-vesicular paracrine signaling. Moreover, it provides a translational based approach to reprogram normal fibroblasts from both uterine and ovarian origin into CAFs using tumor-derived conditioned media. Using these resources, further development of therapeutics that possess potentiality and specificity towards CAF/ECM-mediated chemoresistance in OC are further warranted., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pilar de la Puente and Kristin Calar have a patent for the 3D culture method described in this manuscript, US Patent Application #2022/0228124. Pilar de la Puente is the co-founder of Cellatrix LLC; however, there has been no contribution of the aforementioned entity to the current study. Other authors state no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Sepsis, a life-threatening condition resulting in multiple organ dysfunction, is characterized by a dysregulated immune response to infection. Current treatment options are limited, leading to unsatisfactory outcomes for septic patients. Here, we present a series of studies utilizing compact bone mesenchymal stem cells (CB-MSCs) and their derived paracrine mediators, especially exosome (CB-MSCs-Exo), to treat mice with cecal ligation and puncture-induced sepsis. Our results demonstrate that CB-MSCs treatment significantly improves the survival rate of septic mice by mitigating excessive inflammatory response and attenuating sepsis-induced organ injuries. Furthermore, CB-MSCs-conditioned medium, CB-MSCs secretome (CB-MSCs-Sec), and CB-MSCs-Exo exhibit potent anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated murine macrophage (RAW264.7). Intriguingly, intravenous administration of CB-MSCs-Exo confers superior protection against inflammation and organ damage in septic mice compared to CB-MSCs in certain aspects. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomic analysis, we identify a range of characterized proteins derived from the paracrine activity of CB-MSCs, involved in critical biological processes such as immunomodulation and apoptosis. Our findings highlight that the paracrine products of CB-MSCs could serve as a promising cell-free therapeutic agent for sepsis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) possess a strong ability to modulate the immune response, executed via cytokine-boosted paracrine and direct cell-to-cell contact mechanisms. This reciprocal interaction between immune cells and hPDL-MSCs is influenced by 1,25-dihydroxyvitamin-D 3 (1,25(OH) 2 D 3 ). In this study, the participation of different immunomodulatory mechanisms on the hPDL-MSCs-based effects of 1,25(OH) 2 D 3 on CD4 + T lymphocytes will be elucidated using different co-culture models with various cytokine milieus., Material and Methods: hPDL-MSCs and CD4 + T lymphocytes were co-cultured indirectly and directly with inserts (paracrine interaction only) or directly without inserts (paracrine and direct cell-to-cell contact interaction). They were stimulated with TNF-α or IL-1β in the absence/presence of 1,25(OH) 2 D 3 . After five days of co-cultivation, the CD4 + T lymphocyte proliferation, viability, and cytokine secretion were analyzed. Additionally, the gene expression of soluble and membrane-bound immunomediators was determined in hPDL-MSCs., Results: In the indirect and direct co-culture model with inserts, 1,25(OH) 2 D 3 decreased CD4 + T lymphocyte proliferation and viability. The direct co-culture model without inserts caused the opposite effect. 1,25(OH) 2 D 3 mainly decreased the CD4 + T lymphocyte-associated secretion of cytokines via hPDL-MSCs. The degree of these inhibitions varied between the different co-culture setups. 1,25(OH) 2 D 3 predominantly decreased the expression of the soluble and membrane-bound immunomediators in hPDL-MSCs to a different extent, depending on the co-culture models. The degree of all these effects depended on the absence and presence of exogenous TNF-α and IL-1β., Conclusion: These data assume that 1,25(OH) 2 D 3 differently affects CD4 + T lymphocytes via the paracrine and direct cell-to-cell contact mechanisms of hPDL-MSCs, showing anti- or pro-inflammatory effects depending on the co-culture model type. The local cytokine microenvironment seems to be involved in fine-tuning these effects. Future studies should consider this double-edged observation by executing different co-culture models in parallel., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Behm, Miłek, Schwarz, Rausch-Fan, Moritz and Andrukhov.)

Background: We previously demonstrated that the human amniotic fluid (hAF) from II trimester of gestation is a feasible source of stromal progenitors (human amniotic fluid stem cells, hAFSC), with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic and cardioprotective effects in preclinical models of skeletal and cardiac muscle injury. While hAFSC-EVs isolation can be significantly influenced by in vitro cell culture, here we profiled EVs directly concentrated from hAF as an alternative option and investigated their paracrine potential against oxidative stress., Methods: II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis following written informed consent. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot, and flow cytometry; their metabolic activity was evaluated by oximetric and luminometric analyses and their cargo profiled by proteomics and RNA sequencing. hAF-EV paracrine potential was tested in preclinical in vitro models of oxidative stress and dysfunction on murine C2C12 cells and on 3D human cardiac microtissue., Results: Our protocol resulted in a yield of 6.31 ± 0.98 × 10 9 EVs particles per hAF milliliter showing round cup-shaped morphology and 209.63 ± 6.10 nm average size, with relevant expression of CD81, CD63 and CD9 tetraspanin markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 and able to produce ATP by oxygen consumption. While oxidative stress significantly reduced C2C12 survival, hAF-EV priming resulted in significant rescue of cell viability, with notable recovery of ATP synthesis and concomitant reduction of cell damage and lipid peroxidation activity. 3D human cardiac microtissues treated with hAF-EVs and experiencing H 2 O 2 stress and TGFβ stimulation showed improved survival with a remarkable decrease in the onset of fibrosis., Conclusions: Our results suggest that leftover samples of II trimester human amniotic fluid can represent a feasible source of EVs to counteract oxidative damage on target cells, thus offering a novel candidate therapeutic option to counteract skeletal and cardiac muscle injury., Competing Interests: Declaration of competing interest The authors have nothing to disclose nor competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The regulation of iron metabolism in biological systems centers on providing adequate iron for cellular function while limiting iron toxicity. Because mammals cannot excrete iron, mechanisms have evolved to control iron acquisition, storage, and distribution at both systemic and cellular levels. Hepcidin, the master regulator of iron homeostasis, controls iron flows into plasma through inhibition of the only known mammalian cellular iron exporter ferroportin. Hepcidin is feedback-regulated by iron status and strongly modulated by inflammation and erythropoietic demand. This review highlights recent advances that have changed our understanding of iron metabolism and its regulation.

Mesenchymal Stem Cells (MSCs) have been identified in prostate cancer, raising the critical question of their physical and temporal source. Therefore, MSCs were quantified and characterized in benign and malignant prostate tissue representing different disease states and a wide range of age groups from fetal development through adult death using analytical and functional methodologies. In contrast to lineage-restricted Mesenchymal Progenitor Cells (MPCs) found in normal prostate tissue, MSCs with tri-lineage differentiation potential (adipogenesis, osteogenesis, and chondrogenesis) are identified in prostate tissue from a subset of men with prostate cancer, consistent with an influx of more stem-like progenitors (i.e. MSCs) from the bone marrow. Additionally, prostate tissue from a subset of these patients is highly enriched in MSCs, suggesting their enumeration may have prognostic value for identifying men with aggressive disease. This influx is an ongoing process continuing throughout disease progression as documented by the presence of MSCs in metastatic lesions from multiple organ sites harvested at the time of death in metastatic castration-resistant prostate cancer (mCRPC) patients. This infiltration of MSCs from systemic circulation provides the rationale for their use as a cell-based vector to deliver therapeutic agents.

Thyroid cancer is the most common endocrine malignancy. Although most thyroid cancer patients are successfully treated and have an excellent prognosis, a percentage of these patients will develop aggressive disease and, eventually, progress to anaplastic thyroid cancer. Since most patients with this type of aggressive thyroid carcinoma will die from the disease, new treatment strategies are urgently needed. Tumor cells live in a complex and dynamic tumor microenvironment composed of different types of stromal cells. Cancer-associated fibroblasts (CAFs) are one of the most important cell components in the tumor microenvironment of most solid tumors, including thyroid cancer. CAFs originate mainly from mesenchymal cells and resident fibroblasts that are activated and reprogrammed in response to paracrine factors and cytokines produced and released by tumor cells. Upon reprogramming, which is distinguished by the expression of different marker proteins, CAFs synthesize and secret soluble factors. The secretome of CAFs directly impacts different functions of tumor cells. This bi-directional interplay between CAFs and tumor cells within the tumor microenvironment ends up fostering tumor cancer progression. CAFs are therefore key regulators of tumor progression and represent an under-explored therapeutic target in thyroid cancer.

The role of the local microenvironment in influencing cell behavior is central to both normal development and cancer formation. Here, we show that sprouty 1 (SPRY1) modulates the microenvironment to enable proper mammary branching morphogenesis. This process occurs through negative regulation of epidermal growth factor receptor (EGFR) signaling in mammary stroma. Loss of SPRY1 resulted in up-regulation of EGFR-extracellular signal-regulated kinase (ERK) signaling in response to amphiregulin and transforming growth factor alpha stimulation. Consequently, stromal paracrine signaling and ECM remodeling is augmented, leading to increased epithelial branching in the mutant gland. By contrast, down-regulation of EGFR-ERK signaling due to gain of Sprouty function in the stroma led to stunted epithelial branching. Taken together, our results show that modulation of stromal paracrine signaling and ECM remodeling by SPRY1 regulates mammary epithelial morphogenesis during postnatal development.

Loss-of-function studies have identified Porcupine (PORCN) and Wntless (WLS) as essential mediators of Wnt secretion and signaling. Whereas PORCN is thought to palmitoylate Wnt proteins, WLS is believed to transport palmitoylated Wnt proteins to the cell surface. However, little is known about how these two proteins cooperate to regulate Wnt palmitoylation, trafficking, secretion, and signaling. We first investigated possible interactions between PORCN, WLS, and WNT1, by carrying out co-immunoprecipitation studies. These studies demonstrate the existence of a complex containing PORCN and WLS. They further show that PORCN and WLS compete for binding to WNT1. Then, we used gain-of-function studies to investigate the cooperation between PORCN and WLS as well as possible biochemical interactions between PORCN, WLS, and WNT1. Consistent with the proposed roles for PORCN and WLS, we show that overexpression of PORCN promotes palmitoylation of WNT1 while overexpression of WLS does not. Overexpression of PORCN enhances the ability of WLS to promote WNT1 trafficking to the cell surface as well as secretion, but decreases the ability of WLS to activate WNT1 signaling in target cell. These observations suggest that the levels of WNT1 on the cell surface and in the media are not the sole determinants of the activation of Wnt signaling in target cells.

Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury.

Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.

In periodontitis, gingival fibroblasts (GF) appear to produce a multitude of paracrine factors. However, the influence of GF-derived soluble factors on osteoclastogenesis remains unclear. In this case study, production of paracrine factors by GF was assessed under inflammatory and non-inflammatory conditions, as well as their effect on osteoclastogenesis. Human primary GF were cultured in a transwell system and primed with a cocktail of IL-1β, IL-6 and TNF-α to mimic inflammation. GF were co-cultured directly and indirectly with human peripheral blood mononuclear cells (PBMC). Cytokines and chemokines in supernatants (flow cytometry based multiplex assay), osteoclastogenesis (TRAcP staining) and gene expression (qPCR) were quantified on days 7 and 21. Results from this case study showed that GF communicated via soluble factors with PBMC resulting in a two-fold induction of osteoclasts. Reversely, PBMC induced gene expression of IL-6, OPG and MCP-1 by GF. Remarkably, after priming of GF with cytokines, this communication was impaired and resulted in fewer osteoclasts. This could be partly explained by an increase in IL-10 expression and a decrease in MCP-1 expression. Intriguingly, the short priming of GF resulted in significantly higher expression of inflammatory cytokines that was sustained at both 7 and 21 days. GF appear to produce paracrine factors capable of stimulating osteoclastogenesis in the absence of physical cell-cell interactions. GF cultured in the presence of PBMC or osteoclasts had a remarkably inflammatory phenotype. Given profound expression of both pro- and anti-inflammatory cytokines after the inflammatory stimulus, it is probably the effector hierarchy that leads to fewer osteoclasts., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The mechanism of action of bispecific antibodies (bsAbs) directing T-cell immunity to solid tumors is incompletely understood. Here, we screened a series of CD3xHER2 bsAbs using extracellular matrix (ECM) embedded breast cancer tumoroid arrays exposed to healthy donor-derived T-cells. An initial phase of random T-cell movement throughout the ECM (day 1-2), was followed by a bsAb-dependent phase of active T-cell recruitment to tumoroids (day 2-4), and tumoroid killing (day 4-6). Low affinity HER2 or CD3 arms were compensated for by increasing bsAb concentrations. Instead, a bsAb binding a membrane proximal HER2 epitope supported tumor killing whereas a bsAb binding a membrane distal epitope did not, despite similar affinities and intra-tumoroid localization of the bsAbs, and efficacy in 2D co-cultures. Initial T-cell-tumor contact through effective bsAbs triggered a wave of subsequent T-cell recruitment. This critical surge of T-cell recruitment was explained by paracrine signaling and preceded a full-scale T-cell tumor attack., (© 2024. The Author(s).)